The multi-layered epidermis provides an essential barrier against water loss, physical insults, and infection. Its proper function requires that architectural features be polarized along its entire apical to basal axis (superficial to deep layers). Contributing to the inherent tissue polarity of the epidermis are seven desmosomal cadherins, whose differentiation-dependent arrangement is thought to have functional significance that transcends intercellular adhesion. Indeed, our work during previous funding periods demonstrated that the cadherin desmoglein 1 (Dsg1), which is first expressed as cells commit to differentiate and transit into the suprabasal layers, acts as a scaffold that engages signaling mediators necessary for terminal differentiation. Its failure to be expressed or properly exported to the plasma membrane disrupts organismal homeostasis in patients with Severe dermatitis, Allergies and Metabolic wasting (SAM) syndrome, a syndrome we helped identify in 2013. We hypothesize that Dsg1 coordinates two interrelated but distinct functions required for epidermal morphogenesis: a biochemical program of differentiation, and cytoarchitectural changes required for stratification to form the multi-layered tissue. The resulting tissue provides a protective, dynamic barrier capable of sensing and responding to diverse mechanical and chemical stimuli. The specific objective of this proposal is to determine how Dsg1 mediates changes in tissue architecture and signaling necessary for morphogenesis. To fulfill this objective, we will use gain- and loss-of-function approaches in human 2- and 3D cultures, human patient tissue, and knockout mouse models to address three aims: 1) We will define the machinery that ensures the polarized distribution of Dsg1 in the epidermis, specifically, the extent to which Dsg1 is delivered to the correct position on the plasma membrane through a microtubule minus end-directed dynein-Tctex-Rab3D complex, requiring differentiation-dependent microtubule rearrangements. 2) We will elucidate how Dsg1 modulates the mechanosensitive cortical cytoskeleton to control a temporary drop in tension during formation of the first suprabasal cell layer, and then to concentrate tension in the granular layers later in morphogenesis to ensure proper tight junction structure. 3) We will determine how Dsg1 works with ErbB2 Interacting protein (Erbin) to promote epidermal differentiation by dampening Epidermal Growth Factor Receptor (EGFR) signaling, through disrupting protein complexes permissive for EGFR signaling and/or reducing EGFR mobility within the plasma membrane. These studies will reveal how Dsg1 coordinates cytoarchitectural changes and signaling machinery required for epidermal differentiation, stratification, and barrier formation, and will shed light on how interference with these functions contributes to inherited and acquired skin diseases and cancer.